Our long-term research goal is to produce an engineered osteochondral interface using new hybrid inorganic-organic scaffolds whose gradient in chemical and physical properties make them uniquely capable of inducing a gradual transition from bone- to fibrocartilage-like matrix production by associated human bone marrow-derived mesenchymal stem cells (MSCs). In orthopedic reconstruction, such as that of the anterior cruciate ligament (ACL), soft tissue grafts are often unsuccessful due to poor integration with the associated bone resulting from a failure to reproduce the native-like soft osteochondral interface - a gradual transition from fibrocartilage-like matrix to a bone-like matri. A regenerative strategy to re-establish the osteochondral interface could benefit from recent reports indicating the potent nature of intrinsic scaffold properties in dictating associated cell behavior. In designing scaffolds which promote osteochondral regeneration, two primary challenges exist: (1) the limited knowledge regarding scaffold properties which optimally induce regeneration of bone or fibrocartilage by MSCs and (2) the development of scaffolds with a gradual transition in properties which intrinsically promotes the desired gradual transition in MSC behavior. Given previous literature demonstrating the osteoinductive nature of inorganic, hydrophobic materials, we hypothesized that inorganic-organic hybrid scaffolds could be specifically engineered with gradient chemical and physical properties which would induce a gradual transition in MSC differentiation from bone to fibrocartilage. The proposed gradient scaffolds are based on a combination of inorganic, hydrophobic methacrylated star polydimethylsiloxane (PDMSstar-MA) and organic, hydrophilic poly(ethylene glycol) diacrylate (PEG-DA). The PIs were the first to report the introduction of a PDMS co-macromer into PEG-DA scaffolds and these studies demonstrated that the PDMS co-macromer not only broadens achievable scaffold properties but also modulates cell behavior, including that of MSCs. Fabrication solvents of varying polarities will be used tailor PDMS distribution and porosity. Using existing gradient- making technologies, scaffolds will be prepared as gradients to permit rapid screening of induced MSC behavior. From these results, a inorganic-organic gradient scaffold will be fabricated to regenerate the osteochondral interface in vitro. The specific scope of the present R03 is establishing the feasibility of our hypothesis that these gradient scaffolds' chemical (e.g. inorganic content, chemical functionality, and hydrophilicity) and physical properties (e.g. morphology, porosity, and modulus) will sufficiently induce desired MSC differentiation. The team is comprised of experts in all key areas of the proposed work. Prof. Melissa Grunlan (PI) will lead efforts to fabricate scaffolds. Prof. Mariah Hahn (PI), will lead tissue engineering studies with these scaffolds. Input will be provided by an orthopedic reconstruction specialist, Dr. Walter Lowe (consultant).